DESCRPTION

Technical field

The invention provides novel bacterial strains Pseudomonas jessenii AP3_16 B/00139 and Pseudomonas laurylosulfatovorans AP3_22, which, thanks to produced enzymes, particularly the alkylsulfatases, exhibit exceptional capabilities to survive in high concentrations of surface active agents, such as anionic detergents as well as the ability to decompose such compounds. The invention also provides the method of decontamination of the environment contaminated with sulfuric esters, including surface active agents containing sulfuric-ester ester bond, using microorganism expressing alkylsulfatase, or isolated bacterial alkylsulfatase, wherein said microorganism, its lysate or the isolated enzyme with alkylsulfatase activity is introduced into the treated environment. The invention also provides novel bacterial strains Pseudomonas jessenii AP3_16 B/00139 and Pseudomonas laurylosulfatovorans AP3_22 which are used in said method and/or are the source of the novel proteins with alkylsulfatase activity, as well as composition comprising thereof. The invention also provides novel isolated protein coding alkylsulfatase, the construct of the nucleic acid coding said protein, expression vector able to express nucleic acid construct coding said protein and host cell comprising this nucleid acid construct or the vector which could be used to express this protein. The invention also provides the method of producing novel bacterial strains able to decompose sulfuric esters as well as novel bacterial strains obtained using said method. The invention also provides the method of obtaining the alkylsulfatase protein and preparation comprising it. The invention provides also the use of the novel bacterial strains Pseudomonas jessenii AP3_16 B/00139 and Pseudomonas laurylosulfatovorans AP3_22 and/or lysate thereof, the alkylsulfatase protein, the composition comprising said protein or the novel strain or their mixture for decomposing of the sulfuric esters. Novel bacterial strains as well as novel alkylsulfatases provide effective bioremediation method of environment contaminated with xenobiotics. Novel bacterial strains as well as the purified proteins could be a valuable source of novel biotools useful in the bioremediation of surface active agents, particularly surfactants and detergents or biotransformation in industrial processes. Background art

Surfactants are amphiphilic compounds with both hydrophilic and hydrophobic parts. This allows them to accumulate at the interfaces between air and water, or water and oil phases and lower the surface tension. According to their charge in aqueous solutions, surfactants can be grouped into anionic, nonionic, cationic, or amphoteric classes (Im et al., 2008). The low price and beneficial properties of anionic surfactants make them popular additives to a wide range of products such as: cosmetics, pharmaceuticals, household and industrial cleaning products, and in agriculture as adjuvants improving pesticide spraying properties and penetration.

The extensive application of surface active compounds in household and agricultural products results in accumulation of those compounds in aquatic and soil environments, triggering toxic effects on living organisms. Anionic surfactants such as detergents e.g. lauryl sulfate (SDS, sodium dodecyl sulfate) are known to have bacteriostatic or even bactericidal properties and inhibit the growth of some nitrogen-assimilating cyanobacteria, algae, crustaceans, and also fishes (Lechuga et al., 2016; Sandbacka et al., 2000). Amphoteric features of anionic surfactants underly the mechanism of their accumulation in living organisms and toxicity. Negatively charged acidic part (sulphate group in case of SDS) interact with intracellular components by electrostatic forces, while long chain alcohol binds to cellular proteins by hydrophobic forces (Cserhati et al., 2002).

Problems caused by large amounts of detergents are clearly visible in sewage treatment plants, wherein surface active compounds present in the wastewater negatively influence the physicochemical and biological processes employed in water purification. On the physicochemical level, the decreased surface tension of the seawage causes a deterioration in the flocculation and sedimentation of small particles by stabilizing their colloidal suspension. On the biological level, detergents negatively influence in multiple ways biodiversity of sludge microbial consortium and its role in decomposition of xenobiotics present in seawage. Among the known mechanisms of negative impact several should be mentioned: (I) detergents absorption at the surface of activated sludge floes, triggering bacterial cell lysis; (II) detergents interactions with bacterial cell proteins, causing disruption of their conformation and tertiary structure and in consequence function; (III) detergents binding at enzymes' active sites or substrate-binding pockets disrupting course of chemical reactions (Ivankovic and Hrenovic, 2010). In consequence, surface-active xenobiotics decrease the metabolism of whole groups of microorganisms, by disturbing the activity of certain enzymes, changing the degradation profile of compounds (e.g. carbohydrates) in purified wastewater (Eerlingen et al., 1994). Finally, high surface active agents concentrations in wastewater decrease biodiversity and the metabolic processes conducted by active sludge consortia, making the water purification process inefficient and raising the costs (Zangeneh et al., 2014). Despite known toxicity of the detergents, their use for facilitate bioremediation of hydrocarbon-contaminated soils or water is often proposed in the literature. In this case surfactants are used to increase the water solubility and bioavailability for microorganisms of many hydrophobic xenobiotics (e.g. crude oil and its derivatives, chemical reagents, pesticides, etc.). In this context, lauryl sulfate is one of the most popular detergent proposed for soil bioremediation (Yu et al., 2007; Zhou and Zhu, 2008; Moldes et al., 2013). However, for effective xenobiotics bioremediation stimulated by lauryl sulfate expert suggest use in parallel several types of microorganisms with different biochemical properties. In the first order it is proposed to use microorganisms capable of degrading xenobiotics but resistant to high concentrations of detergent. Next to use of microorganisms effectively removing surface active agents from environment, when the xenobiotic bioremediation process was completed.

There are various reports describing the isolation of bacterial strains having the ability of surface active agents degradation, including degradation of sodium lauryl sulfate. Among the described SDS-degrading microorganisms are Pseudomonas strains (Chaturvedi and Kumar, 201 1 a; John et al., 2015; Klebensberger et al., 2006; Yeldho et al., 201 1 ), Kliebsiella oxytoca (Masdor et al., 2015; Shukor et al., 2009), Enterobacter sp. strain NENI-13 (Rahman et al. 2016), and finally microbial consortia composed of Acinetobacter calcoaceticus and Pantoea agglomerans (Abboud et al., 2007). However, in above mentioned examples the microorganisms were selected from environments highly contaminated solely with detergents where there were no other xenobiotics present.

Surface active agents such as detergents are also widely used as an additives to products containing pesticides allowing the final product to fullfil demands of the end user.

Enzymes hydrolysing sulfur esters, in particular anionic detergents

The degradation process of anionic surface active agents or sulfuric acid esters of alkyl or aromatic alcohols has been well characterized at the biochemical level. Enzymes called sulfatases (EC 3.1.6.-) are involved in the hydrolisys of sulfate ester bonds (usually leading to the formation of the appropriate alcohol and inorganic sulphate). This heterogeneous group of enzymes can be divided into at least three distinct classes, depending on the mechanism of catalysis (Kertesz, 2000).

The first group gathers arylsulfatases, proteins with highly conserved consensus motif

C/S-X-P-X-A-X4-T-G, identified mostly in eukaryotic organisms (Hagelueken et al., 2006). Although, the class name suggests that these enzymes are specific for aromatic compounds, many of them also catalyze desulfurization of sulfated alifatic carbohydrates (Toesch et al., 201 ). The AtsA enzyme from Pseudomonas aeruginosa is a bacterial representative of this group (Boltes et al., 2001). The second group consists of Fe(ll) dependent dioxygenases, catalyzing an oxidative cleavage of the sulfate ester to the corresponding aldehyde and inorganic sulfate, with AtsK from Pseudomonas putida as a model example (Miiller et al., 2004). The third class includes enzymes with the metallo-3-lactamase-like domain at the N-terminus and SCP-2-like domain at C-terminus of the protein, involved in the binding of sterols. Curently, this sulfatase class is represented by four sulfatases with different substrate specificity: SdsA having activity towards sodium dodecyl sulfate (SDS) (Davison et al., 1992), SdsAP and SdsA1 active toward primary sulfate esters including sodium dodecyl sulfate (Hagelueken et al., 2006; Long et al., 201 1 ; Schober et al., 201 1 ) and PisA1 specific towards secondary alkyl sulfates (Schober et al., 201 1 ). Enzymes from this group differ in the mechanism of action and have ability to catalyze cleavage either of S-0 or C-0 bond causing, in case of assymetrical substrate, the retention or inversion of the product alcohol, which could be verified using chiral or labeled substrates. Early research for SdsA1 and PisA1 proteins revealed that both sulfatases catalyze cleavage of the C-0 bond.

Disclosure of the invention

In the light of the described state of art, the aim of the present invention is to overcome the inconveniences related with removal of anionic detergents from the environment for example from soil, water, solution, preferably wastewater, industrial installation. The invention is also intended to provide tools to catalyze the chemical reactions leading to hydrolysis of alkyl and/or arylo sulfonic derivatives.

The object of this invention is therefore to provide novel bacterial strains and/or constructs to obtain improved bacterial strains able to catalyze the hydrolysis of sulfuric esters, e.g. surface active agents, which are also resistant to high concentrations of surface active agents in the environment and other xenobiotics, e.g. pesticides or hydrocarbons. The object of this invention is also to provide novel alkylsulfatases, production of which in the active form allowing to catalyze the hydrolysis of the ester bond of sulfuric esters, e.g. surface active agents, what is possible by their overexpression in the heterologous expression systems preferably Eschericha coli host cells. The object of this invention is also to provide alkylsulfatases preparations which are able to hydrolyze the selected sulfuric esters, e.g. surface active agents.

The microorganisms described in the present patent application were isolated from the peaty soil (originating from the root zone) sampled from the subsurface flow constructed wetland of a wastewater treatment plant operated by pesticide packaging company. This strategy allowed to isolate microorganisms capable of both degradation of surface active agents as well as having resistance to high concentrations of other xenobiotics, e.g. pesticides. Moreover, the experiments also revealed in the isolated microorganisms presence of enzymes with alkylsulfatase activity, which are able to catalyze the cleavage of the sodium dodecyl sulfate to the sulfiric acid residue and the long-chain alcohol. Enzymes with sulfatases activity isolated from the bacterial strains described in present invention were characterized regarding the amino acid composition and biochemical properties. The results showed that the proteins should be classified to the third class of bacterial sulfatases. The inventors isolated and characterized 36 bacterial strains of Pseudomonas spp. that are able to decompose detergent - sodium dodecyl sulfate, although they varied significantly in their ability to use this surface active agent as the sole carbon source. The microorganisms were isolated from the subsurface flow constructed wetland of a wastewater treatment plant operated by pesticide packaging company. The isolated microorganisms showed the taxonomical similarity to Pseudomonas jessenii subgroup. In biochemical tests they showed the best SDS degradation capabilities as they were able to decompose from 80% to 100% of the SDS present in culture in an initial concentration 1 g/L in less than 24 h. These strains differ significantly in SDS degradation rate, the resistance to high detergent concentration (ranging from 2.5 g/L to 10 g/L and even higher) as well as in the chemotaxis towards SDS on a plate test. Zymography assay of the cytoplasmatic protein fraction of the five most efficiently SDS degrading strains revealed in all of the selected isolates presence of proteins with alkylsulfatase activity. For two of these proteins encoded in the genomes of selected strains their activity towards sodium dodecyl sulfate was experimentally confirmed and the proteins were overexpressed and purified for further characterization.

The obtained results showed that the isolated microorganisms and the purified proteins preparations could be a valuable source of biotechnological tools useful in the bioremediation of surface active agents or biotransformation in industrial processes.

The invention also provides the method of decontamination of the environment, in particular soil, water, solution, industrial installation contaminated with sulfuric esters, in particular surface active agents including compounds containing sulfuric-ester ester bond, using microorganism expressing alkylsulfatase, or isolated bacterial alkylsulfatase wherein

alkylsulfatase comprises amino acid sequence encoded by nucleotide sequence comprising sequence set forth in SEQ ID NO: 3 or 4 or at least 95% identical to it, in particular alkylsulfatase comprises amino acid sequence set forth in SEQ ID NO: 1 or 2 or at least 95% identical to it;

wherein aforementioned microorganism, its lysate or the enzyme with alkylsulfatase activity isolated from the microorganism is introduced into the treated environment.

In the preferred embodiment of the method of decontamination of the environment contaminated with sulfuric esters the sulfuric ester is sodium dodecyl sulfate.

The object of the invention is also the Pseudomonas jessenii AP3_16 strain deposited on the 30 ^th August 2017 in the Polish Collection of Microorganisms (PCM) of the Institute of Immunology and Experimental Therapy PAS under the number B/00139.

The object of the invention is also the Pseudomonas laurylsulfatovorans AP3_22 strain deposited on the 30 ^th August 2017 in the Polish Collection of Microorganisms (PCM) of the Institute of Immunology and Experimental Therapy PAS under the number B/00140. The deposited bacterial strains of the invention show the expression of the alkylsulfatases comprising amino acid sequences set forth in SEQ ID NO: 1 and 2 (for Pseudomonas laurylsulfatovorans AP3_22 and Pseudomonas jessenii AP3_16 respectively). The alkylsulfatases expressed by the aforementioned strains show high sequence similarity (about 93%) and ensure efficient degradation of sulfuric esters, in particular surface active agents including compounds containing sulfuric-ester ester bond, in particular sodium dodecyl sulfate. Moreover, the inventors reported that the alkylsulfatase comprising the sequence set forth in SEQ ID NO: 1 and 2 could be introduced to other bacterial species and enabled these species to efficiently degrade aforementioned surface active agents. These enzymes are also able to hydrolyze the ester bond of the sulfuric esters including surface active agents comprising such bond, e.g. sodium dodecyl sulfate, in broad range of pH and temperature. Moreover, the inventors stated that both specified Pseudomonas strains show resistance to xenobiotics and could be used in the bioremediation of environment contaminated not only by surface active agents but also other xenobiotics, e.g. pesticides or hydrocarbons.

Therefore, the object of the invention is also the method of the environment decontamination, wherein the treatment of the environment contaminated with sulfuric esters including surface active agents including compounds containing sulfuric-ester ester bond is a part of bioremediation process of the environment contaminated with xenobiotics, preferably hydrocarbons wherein the microorganism producing alkylsulfatase is Pseudomonas jessenii AP3_16 and/or Pseudomonas laurylsulfatovorans AP3_22.

The object of the invention is also protein, comprising amino acid sequence encoded by nucleotide sequence comprising sequence set forth in SEQ ID NO: 3 or 4 or at least 95% identical with it, in particular comprising amino acid sequence set forth in SEQ ID NO: 1 or 2 or sequence at least 95% identical with it, with alkylsulfatase activity.

The object of the invention is also the nucleic acid construct encoding protein according to the invention.

The object of the invention is also an expression vector comprising the construct according to the invention.

The object of the invention is also the host cell comprising construct according to the invention or the expression vector according to the invention.

In the preferred embodiment, the host cell is a single-cell mesophilic organism, in particular E. coli, more preferably E. coli BL21.

The object of the invention is also the method of producing the bacterial strains able to decompose sulfuric esters including surface active agents comprising compounds containing sulfuric-ester ester bond in environment, comprising introducting of the construct according to the invention or vector according to the invention into the bacterial cell by genetic engineering or using natural horizontal gene transfer, preferably directly in the environment.

The object of the invention is also a bacterial strain obtained by the method of producing bacterial strains able to decompose sulfuric esters including surface active agents comprising compounds containing sulfuric-ester ester bond according to the invention.

The object of the invention is also the method of production of alkylsulfatase protein, comprising the step of culturing the host cell in conditions allowing the expression of proteins, wherein the host cell expresses the protein according to the invention comprising amino acid sequence encoded by nucleotide sequence comprising sequence set forth in SEQ ID NO: 3 or 4 or at least 95% identical with it, in particular protein comprising amino acid sequence set forth in SEQ ID NO: 1 or 2 or sequence at least 95% identical with it with alkylsulfatase activity.

The object of the invention is also the composition comprising the protein according to the invention or protein produced with the method according to the invention.

The object of the invention is also the composition comprising the bacterial strain Pseudomonas jessenii AP3_16 and/or Pseudomonas laurylsulfatovorans AP3_22 and/or bacterial strain obtained by the method of providing the bacterial strains able to decompose sulfuric esters according to the invention and/or the cell comprising the construct according to the invention and/or the vector according to the invention.

The object of the invention is also the use of the bacterial strain Pseudomonas jessenii AP3_16 and/or Pseudomonas laurylsulfatovorans AP3_22 and/or bacterial strain obtained by the method of providing the bacterial strains able to decompose sulfuric esters according to the invention and/or the lysate of the aforementioned strains or the protein according to the invention and/or the composition comprising the bacterial strain Pseudomonas jessenii AP3_16 and/or Pseudomonas laurylsulfatovorans AP3_22 and/or bacterial strain obtained by the method of providing the bacterial strains able to decompose sulfuric esters according to the invention and/or the cell comprising the construct according to the invention and/or the vector according to the invention to the decomposition of the sulfuric esters including surface active agents comprising compounds containing sulfuric-ester ester bond in the environment, in particular soil, water, solution, industrial installation.

The object of the invention is also the method of bioremediation of environment contaminated with xenobiotics comprising:

a) step of introducting of the surface active agents comprising compounds containing sulfuric ester ester bond to increase the solubility of hydrocarbons in the water solutions and thus improve their bioavailability for microorganisms b) step of introducting of the microorganisms capable of degrading the xenobiotic which caused the contamination but also resistant to the high concentrations of surface active agents, and

c) step of introducting of the novel bacterial strain Pseudomonas jessenii AP3_16 and/or Pseudomonas laurylsulfatovorans AP3_22 and/or bacterial strain obtained by the method of providing the bacterial strains able to decompose sulfuric esters according to the invention and/or the lysate of the aforementioned strains or the protein according to the invention and/or the composition comprising strains according to the invention or a combination thereof, to remove surface active agents from the treated water or soil.

Scope of the present invention covers novel strains Pseudomonas jessenii AP3_16 deposited on the 30 ^th August 2017 in the Polish Collection of Microorganisms (PCM) of the Institute of Immunology and Experimental Therapy PAS under the number B/00139 and Pseudomonas laurylosulfatovorans AP3_22 deposited on the 30 ^th August 2017 in the Polish Collection of Microorganisms (PCM) of the Institute of Immunology and Experimental Therapy PAS under the number B/00140 and their functional derivatives.

The term novel variant of the strain or strains obtained by the method according to the invention or functional derivative of the strain is to be understood as a mutant strain or strain obtained by culturing the deposited strain or strains obtained by the method according to the invention as the starting material, which comprises the nucleic acid fragment coding at least one of the alkylsulfatases set forth in the SEQ ID NO: 1-2, in particular nucleic acid fragment comprising sequence set forth in SEQ ID NO: 3 or 4, wherein such strain is able to hydrolyze the ester bond of the sulfuric esters, in particular surface active agents.

The inventors reported that strains belonging to Pseudomonas jessenii group, preferably Pseudomonas jessenii AP3_16 and Pseudomonas laurylsulfatovorans AP3_22 are able to survive in the highly contaminated environment and also have the ability to decompose surface active agents, preferably sodium dodecyl sulfate up to 80% from the initial concentration of 1 g/L within 8 hours and are able to tolerate very high concentration of SDS in the environment reaching up to 25 g/L.

The inventors, basing on the sequence similarity, identified in the genomes of the isolated strains of Pseudomonas spp. able to hydrolyze the ester bond of sulfuric esters genes encoding sulfatases. Twenty one genome fragments comprising identified genes together with the promoter regions were cloned into vectors maintained in E. coli TOP10 strain, which were then tested for the SDS degradation capabilities. Unexpectedly, two of the obtained constructs provide the host cells with the ability to decompose sodium dodecyl sulfate.

Such strains or their lysates or proteins isolated from these strains are useful in the bioremediation including the bioremediation of environments contaminated with surface active agents. The constructs, e.g. plasmids carrying the genes providing the strains the abilities to hydrolyze the ester bond of sulfuric esters including surface active agents such as sodium dodecyl sulfate could be used for the production of novel strains capable of this process or to improve the strains already having such properties.

Term„bioremediation" is to be understood as transformation of the harmful substances present in the environment to the less toxic or completely safe metabolites using the microorganisms or higher organisms.

According to the invention, „bioaugmentation" means the introduction into natural or degraded environment, of selected strains or a composition of microorganisms in order to increase the performance and capabilities of the course of a given process.

In case there is no possibility of directly constructing strains able to decompose the surface active agent based on the indigenous microflora, the plasmid can be introduced into the environment through the method of bioaugmentation. A strain comprising construct or vector carrying any nucleotide sequence set forth in SEQ ID NO: 3-4 or their derivatives is introduced into the environment contaminated with surface active agents and as a result of natural horizontal gene transfer, e.g. the conjugation, the construct or vector comprising any nucleotide sequence set forth in SEQ ID NO 3-4 according to the invention is transferred into the cells of indigenous microflora.

The inventors developed the method of the construction of strains able to hydrolyze the ester bond of the sulfuric esters, in particular surface active agents, also based on the autochthonic microorganisms isolated from the natural environment, without geographical limitations, preferably from the environment contaminated with surface active agents or hydrocarbons or pesticides mobilization and decontamination of which require usage of surface active agents.

Thanks to the present solution it is possible to remove surface active agents comprising ester bond by the hydrolysis of the ester bond of sulfuric esters. For example, the hydrolysis of S-0 bond is carried out by the Pseudomonas jessenii AP3_16 or Pseudomonas laurylsulfatovorans AP3_22 or novel bacterial strain able to hydrolyze the ester bond of sulfuric esters obtained by the method according to the invention or the composition of strains able to hydrolyze the ester bond of the sulfuric esters or combination thereof.

In another aspect the invention relates to the construct or vector comprising any nucleotide sequence set forth in SEQ ID NO: 3-4 or its functional derivative. Such construct could be used as a plasmid or as a sequence fragment integrated into bacterial genome for constructing strains capable of hydrolysis of esters of aliphatic and aromatic alcohols, in particular surface active agents. For removing the contamination with surface active agents a bacterial strain comprising construct or vector, for example a plasmid comprising any nucleotide sequence set forth in SEQ ID NO: 3-4 or its functional derivative or bacterial strain comprising any nucleotide sequence set forth in SEQ ID NO: 3-4 or its functional derivative integrated into its genome could be used. Strains comprising any nucleotide sequence set forth in SEQ ID NO: 3-4 or its functional derivative would be able to hydrolyze the esters of aliphatic and aromatic alcohols.

The term„alkylsulfatase" refers to the protein - an enzyme - belonging to the sulfatase group involved in the hydrolysis of the ester bond of sulfuric esters (which usually leads to the formation of inorganic sulfate and corresponding alcohol). Term substrate is to be understood as any organic compound comprising the C-O or S-0 bond.

The inventors reported that alkylsulfatases such as CD175_09595 (SEQ ID NO: 2) or B0D71_15760 (SEQ ID NO: 1) could be used in the hydrolysis of ester bond of sulfuric esters. Particularly preferred application is to use these proteins in the hydrolysis of the ester bond of the sulfuric esters in solutions containing K ⁺ and Mg ²⁺ ions which increase activity of these sulfatases. However the analyzed alkylsufatases differ in the sensitivity towards Na ⁺ , Mn ²⁺ and 10 mM Ca ²⁺ ions. B0D71_15760 (SEQ ID NO: 1 ) enzyme is activated with Na ⁺ ions in 10 mM concentration, although higer concentration of these ions (100 mM) have no significant effect on the alkylsulfatase activity. On the other hand the CD175_09595 (SEQ ID NO: 2) is inhibited by the highier concentrations of Na ⁺ ions, while lower sodium ions concentration have no effect on enzyme activity. Mn ²⁺ ions did not affect CD175_09595 (SEQ ID NO: 2) activity, although they have positive effect on B0D71_15670 (SEQ ID NO: 1 ) activity, similarly to lower tested concentration of 10mM Ca ²⁺ ions.

The use of alkylsulfatase such as CD175_09595 (SEQ ID NO: 2) or B0D71_15760 (SEQ ID NO: 1) in the hydrolysis of ester bond of sulfuric esters could be performed in the pH of which is preferably in pH range from 6.0 to 9.0 with optimum in pH 8 and in broad spectrum of tempertures (30-90°C). The optimal temperature for CD175_09595 (SEQ ID NO: 2) alkylsulfatase is 60°C and for B0D71_15760 (SEQ ID NO: 1 ) alkylsulfatase it is 70°C.

The inventors reported that the use of B0D71_15760 (SEQ ID NO: 1 ) alkylsulfatase in the hydrolysis of the ester bond of sulfuric esters could be performed even during one hour incubation at the optimal temperature of 60°C.

It is also possible to use the immobilized forms of alkylsulfatase, preferably absorbed on the support or onto a carrier matrix of both inorganic (e.g. glass, silica, metal oxide) and organic (e.g. proteins, polysaccharides, synthetic polymers) type. Alkylsulfatases could be also immobilized using for example cross-linking or enclosure within semipermeable membranes. Brief description of drawings For a better understanding of the invention, it has been ilustrated in the embodiments and in the accompanying figures, in which:

FIGURE 1. Shows the identification of bacteria strains potentially degrading SDS. The photographs show a comparison of growth profile of selected strains cultured on (A) agar solidified 0.1X LB medium or (B) basal medium supplemented with 0.1 % SDS.

Differences in clear zone diameter observed for each strain cultured on the medium containing SDS as the sole carbon source indicate a differing abilities of detergent degradation.

FIGURE 2. Shows a study of the growth rate and SDS degradation ability of isolated bacterial strains. (A) Correlation of growth rate (increase in OD) and SDS degradation ability (decrease in substrate concentration [%]) after 24 h of incubation. The dotted line represents correlation plotted for the 36 selected strains. (B) Time course study of SDS degradation rate of the five most effective bacterial strains. (C) Time course study of growth rate for the five most effective bacterial strains in liquid cultures containing 0.1 % SDS as the sole carbon source. The values are mean values of the three replicates, and the error bars indicate the standard deviations.

FIGURE 3. Shows a phylogenetic analysis of selected bacterial isolates based on the 16s rRNA sequence. The phylogenetic analysis was computed by Maximum Likelihood method. Examined isolates are marked with black triangles.

FIGURE 4. Shows the effect of SDS concentration on the bacterial growth rates of selected bacterial strains reflected as optical density of cell cultures after 24 h of incubation. The values are mean values of the three replicates, and the error bars indicate the standard deviations. The dotted line represents the initial OD ₆₀ o of each strain.

FIGURE 5. Shows an assay for bacterial chemotaxis towards SDS being the only source of carbon. Bacterial growth was observed as rings formed around a source of carbon formed after 8 h of incubation at 30°C. Rows represent selected strains' chemotactic response toward glucose (I), SDS (II) and control plates without carbon source (III).

FIGURE 6. Shows an alkylsulfatase activity analysis in bacterial lysates of five strains separated in 8% native polyacryloamide gel. The strains were cultured on minimal medium in the presence of glucose (MM + glucose) or SDS (MM + SDS) as the only carbon sources. The presence of insoluble lauryl alcohol bands demonstrating the presence of SDS-degrading proteins was identified after 30 min. or 4 h of incubation.

FIGURE 7. Shows a comparison of degradation capabilities of E. coli and new strains carrying vectors with different genome fragments coding the potential alkylsulfatases identified in the genomes of AP3_16 and AP3_22 strains. The strains were cultured in LB medium supplemented with 0.1 % SDS. FIGURE 8. Shows the effect of (A) pH, and (B) temperature on activity of the homogenous protein preparations. The activity of the protein preparations was tested (A) in pH range of 4-10 or (B) in the temperature range of 4-90 ^° C by determining SDS concentration changes in the solutions using the Stains-all reagent. The values marked with squares were obtained for the CD175_09595 protein and marked with triangles were obtained for the B0D71_15760 protein.The detergent concentration was measured after 24 h using Stains-all reagent.

Description of embodiments

The following examples are presented merely to illustrate the invention and to clarify its various aspects, but are not intended to be limitative, and should not be equated with all its scope, which is defined in the appended claims.

In the following examples, unless it was otherwise indicated, standard materials and methods described in Sambrook J. and Russel D. W. 2001 Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York were used, or the manufacturers' instructions for specific materials and methods were followed.

Examples

Example 1. Selection and identification of bacteria capable of degradation of anionic surface active agents

10 g of the peaty soil collected from biological wastewater treatment plant stored in a plastic bag at 8°C were placed into the sterile container of a Waring blender and 100 mL of 0.9% NaCI was added. The sample was homogenized by three blending cycles at maximum speed for 1 minute, with intermittent cooling on ice for 1 minute. After 5 minutes of sedimentation, the liquid fraction was transferred into a sterile beaker. Another 100 mL of 0.9% NaCI was then added to the soil sludge, and the whole procedure was repeated three times in total. All the fractions were pooled together and centrifuged at 5,000 g for ten minutes at 4°C, and then pellet comprising mineral residues and soil bacteria was collected. Several dilutions of the obtained pellet were plated on solid soil extracts plates. After 7 days of incubation at 23°C, colonies with unique morphologies were transferred into 96-well plates. Colonies were banked in 100 [iL of liquid soil extract supplemented with 15% glycerol and stored at -80°C. The isolation procedure based on the ability to grow on the solid soil extract under laboratory conditions resulted in the selection of 238 morphologically distinctive colonies.

Isolated microorganisms were replicated on solid basal medium plates (per liter: 3.5 g KH ₂ P0 ₄ ; 1.5 g K ₂ HP0 ₄ ; 0.25 g (NH ₄ ) ₂ S0 ₄ ; 0.5 g NaCI; 0.14 g MgS0 ₄ ; 0.15 g MgCI ₂ x6H ₂ 0) (Shahbazi et al., 2013) with SDS (2 g/L) as the carbon source to test the ability of sodium lauryl sulfate degradation. In the basal medium detergent can precipitate in the form of a homogeneous suspension, which facilitates the observation of clear zone that arise around the growing bacterial colony. After 3-4 days of incubation at 30°C, isolates with clear halos around the colonies were considered to be able to degrade SDS (Fig. 1). For 36 isolates, a clear halo surrounding the colony was observed. In addition, the purity and homogeneity of the isolates was verified by two independent streaking passages on the same medium.

Example 2. Determination of the ability of isolated strains to degrade anionic surfactants

The ability to degrade sodium lauryl sulfate by 36 bacterial strains showing clear halo surrounding the colony on solid substrates was quantified using colorimetric methods.

The selected isolates were pre-cultured overnight in liquid 0.1X LB medium (10 times diluted LB medium) unsupplemented with detergent with 140 rpm orbital agitation. The overnight culture was centrifuged and washed with minimal medium (per liter: 0.5 g Na ₂ HP0 ₄ ; 0.5 g KH ₂ P0 ₄ ; 0.25 g (NH ) ₂ S0 ₄ ) without any carbon source and then used to inoculate liquid minimal medium supplemented with 0.1 % SDS (1 g/L) to OD ₆₀ o=0.05-0.15. The detergent concentration was measured by colorimetric assay at 0, 2, 4, 6, 8, and 24 hours after the beginning of the experiment. The minimal medium was used in liquid cultures for colorimetric assays because there was no SDS precipitation observed which could negatively influence measurements.

The SDS concentration was determined in a 96-well plate using Stains-All reagent (Sigma-Aldrich) as previously described (Rusconi et al., 2001) with minor modifications. Stains- All stock solution (1 mg/mL) was prepared in isopropanohwater (50:50). The working solution consisted of 1 mL of the stock diluted in 18 mL of water and 1 mL of formamide. Samples from each time points were centrifuged and the clear, bacteria-free supernatant was diluted diluted 10 times in water. 8 pL of diluted sample were dispensed into the well, supplemented with 100 pL of water and 100 pL of the Stains-All working solution, and finally the absorbance at 438 nm was measured after 10 minutes of incubation at RT. The measurements were carried out in triplicate. Results of colorimetric assay were confirmed also by mass spectrometry.

Two out of the 36 bacterial strains did not adapt to the liquid culture conditions, which resulted in a minimal increase in the optical density of the culture with no SDS decomposition after 24 hours of incubation. In the remaining 34 cases, a decrease in the initial detergent concentration was observed ranging from 6.4% to 99.2% (Fig. 2A). The isolates analyzed in this way were arranged into four groups reflecting their sodium lauryl sulfate degradation ability:

• non-degraders - 2 isolates that did not adapt to the culture conditions,

• slow-degraders - 14 isolates, with an degree of SDS degradation below 30% within 24 hours,

• medium-degraders - 15 isolates, with a degree of SDS degradation between 31 % and 70% within 24 hours ^• fast-degraders - 5 isolates with the degree of SDS degradation exceeding 70%.

The last, most promising group of microorganisms is strain AP3_22, which has degraded about 50% and 81 % of the initial amount of sodium lauryl sulfate at 6 and 8 hours, respectively (Fig. 2B). After 24 hours of the experiment with the colorimetric measurement, the fast degrading isolates were able to degrade: 74.1 % - AP3_10; 84.6% - AP3_16; 85.1 % - AP3_19, 79.4% - AP3_20 and 99.2% - AP3_22 of detergent from the initial concentration of 1 g/L. This experiment also showed a strong positive correlation between the degree of SDS degradation and the increase in the optical density of the bacterial culture (correlation coefficient r = 0.86) (Fig. 2A). Isolates AP3_16 and AP3_22 have very similar growth pattern (Fig. 2C) but they differ significantly in SDS degradation rate (Fig. 2B).

Example 3. Taxonomic identification of strains degrading anionic surface active agents, including the five most efficient strains.

Taxonomic analysis of 36 isolated strains was based on Sanger's sequencing of the V3- V4 region of the 16S rRNA gene amplicons (approximately 1 ,500 bp).

Small subunit (16S) of the rRNA gene was amplified by colony PCR using the following primer pair: 27F (AGAGTTTGATCCTGGCTCAG) SEQ ID NR: 7 and 1492R (GGTTACCTTGTTACGACTT) SEQ ID NR: 8 (Lane, 1991). The PCR reaction was carried out in a volume of 50 μ!_ on DNA template of a given strain using Phusion High-Fidelity DNA Polymerase (Thermo) in the presence of HF Buffer. The PCR involved: one cycle at 99°C for 5 min; 10 cycles at 99°C for 30 s, 60°C for 30 s, and 72°C for 45 s; and 20 cycles at 99°C for 30 s, 50°C for 30 s, and 72°C for 45 s with final elongation at 72°C for 5 min. The PCR products (approximately 1 ,500 bp) of 16S rRNA genes were purified using AMPureXP with a 0.8: 1 ratio, then cloned into a pCR™Blunt ll-TOPO® vector (ThermoFisher) according to the manufacturer's protocol, and transformed to Escherichia coli (Sambrook and Russel, 2001). The plasmids were isolated using a Plasmid Mini kit (A&A Biotechnology) and the inserts - 16S rRNA gene fragments were sequenced with Sanger using universal M 13 Forward (GTAAAACGACGGCCAG) SEQ ID NR: 9 and M13 Reverse (CAGGAAACAGCTATGAC) SEQ ID NR: 10 primers (ThermoFisher). 16S rRNA partial gene sequences were compared to the EzBioCloud database (Yoon et al., 2017). The general phylogenetic anlysis showed that selected 36 bacterial strains belong to the genus Pseudomonas.

The 16S rRNA sequence for Pseudomonas jessenii AP3_16 deposited under No. B/00139 is presented as SEQ ID NO: 5 and for Pseudomonas laurylosulfatovorans AP3_22 deposited under No. B/00140 as SEQ ID NO: 6.

The sequences were aligned to each other using ClustalW (Larkin et al., 2007). 16S rRNA gene sequences of the five selected SDS-degrading isolates and 27 representative sequences of reference type strains from the NCBI database were used to construct a phylogenetic tree. Multiple sequence alignment to identify the maximum identical gene fragment was carried out using ClustalW (Larkin et al., 2007). A maximum likelihood tree was constructed in MEGA 6.0 (Tamura et al., 2013) with 1 ,000 bootstrap replicates using Tamura-Nei model and default settings.

Detailed analysis of the 16S rRNA amplicons sequence alignment of the five best degradative strains indicated that the isolates were highly similar to each other but not identical (Fig. 3). The comparison of these sequences to the EzBioCloud database revealed that the four of the fast-degrader isolates showed the highest similarity (AP3_10 - 99.79%, AP3_16 - 99.24%, AP3_20 - 99.86% and AP3_22 - 99.79%) to P. jessenii CIP 105274. However, the AP3_19 isolate shared the highest similarity (99.45%) with P. mohnii lpa-2. Analysis of phylogenetic tree (Fig 3) shows that the entire SDS-degrading group falls within a clade. AP3_10, AP3_16 and AP3_22 isolates clustered with P. jessenii CIP 1052274, P. reinekei CCUG 531 16, P. koreensis LMG 21318, and P. moraviensis DSM 16007, while AP3_16, and AP3_19 were sister taxa that clustered together in a group that included the previously mentioned clade, plus P. moorei CCUG 53114, and P. vancouverensis DSM 17555. Above taxonomic analysis indicate that the isolated microorganisms belongs to the Pseudomonas family, with the five best degrading isolates belonging to the Pseudomonas jessenii subgroup.

Example 4 - Determination of optimal growth conditions and maximum tolerated concentration of surface active agents for new bacterial strains

The selected isolates were pre-cultured overnight in LB medium, centrifuged, washed two times with minimal medium without any carbon source, and then inoculated in minimal medium to initial optital density OD ₆₀ o = 0.15 with different SDS concentrations (0.5 - 50 g/L) as the sole carbon source. The isolates were cultured in triplicate in deep 48-well plates. The optical density of the cultures (OD ₆₀₀ ) was monitored after 24 hours of incubation. Statistical analysis and interpretation of obtained data was made using the R package version 3.5.0. The Kruskal- Wallis with the Dunn's post hoc tests were used to test differences between initial and final optical density of cultures, with significance set to 0.05.

Bacterial growth, expressed as an increase of optical density (above the initial OD ₆₀ c=0.15), was interpreted as a positive result showing that under given detergent concentration, the bacteria are able to use SDS as a carbon source (Fig. 4), which allows cell growth and division. All effective degrader isolates reached their highest optical density (OD ₆₀₀ =0.55-0.75) in the presence of 1 g/L SDS, which could be interpreted as a sign of both a sufficient availability of the carbon source and the minimal negative effects of the detergent. The first SDS toxicity was observed in cultures supplemented by 2.5 g/L of SDS, where the optical density (OD ₆₀₀ ) for all five isolates of fast-degraders was similar or even below the levels observed for 0.5g /L SDS supplemented samples. Presence of 5 g/L of detergent in medium had a significant toxic effects on the AP3_10 isolate, as the optical density of this culture did not change after 24 hours (p-value=0.76). Meanwhile, for the other four isolates, significantly increased, as their cultures reached an optical density from 0.22 to 0.4 (p-value <0.05). In the presence of 10 g/L of SDS, only isolate AP3_16 retained an ability to grow (p-value=0.02). An intriguing behavior was observed for isolate AP3_16, which in contrast to the other isolates retained an OD ₆₀ o of 0.12 in the presence of 20 g/L and 25 g/L of detergent which was statistically not important change from initial OD ₆₀₀ =0.15 (p-value=0.45). Such result suggests that this strain maintained the ability at least to survive in the presence of high SDS concentrations.

Example 5 - Chemotaxis of isolated bacterial strains towards a source of sodium lauryl sulfate.

The chemotactic response of the five selected isolates toward SDS was studied by a drop plate assay, with glucose as a positive control. Overnight LB-pre-cultures of the selected isolates AP3_10, AP3_16, AP3_19, AP3_20, oraz AP3_22 were used to inoculate fresh 50 mL LB medium (1 :50, v/v). After 2 hours of incubation at 30°C, cells were harvested by centrifugation at 8,000 rpm for 15 minutes, washed twice with 0.9% NaCI, resuspended to a final concentration OD ₆ oo=0.1 in minimal medium (temp. 40°C) containing 0.45% bacto agar and poured into 30 mm Petri plates. Next five pL of 20% SDS or 20% glucose were dropped into the center of the petri plate, and the chemotactic response (Fig. 5) was observed after 8 h of incubation at 30°C. On the positive control plates, each isolate migrated up to the center of the dish where the glucose was dropped, whereas on the plates with SDS the growth of bacteria as a concentric rings indicated that the strain was growing in the area of optimal detergent concentration.

Example 6 - Morphological, physiological and biochemical analysis of bacterial strains with high ability to degrade anionic surface active agents

Gram staining was carried out by standard methods. Cell morphology was analyzed by light microscopy and transmission electron microscopy after overnight cultivation in LB medium at 30°C. Flagella staining was performed using a wet-mount technique with Ryu stain (Heimbrook i wsp., 1989). Temperature, pH and salinity tolerance of the strains were analyzed by monitoring changes in optical density (OD ₆₀ o) in liquid cultures (in comparison to non- inoculated controls). Overnight cultures were diluted in fresh LB media with adjustments for the following assays: pH 7 for temperature assay (4°C, 8°C, 15°C, 23°C, 30°C, 37°C, 42°C); pH 3.0-11.0 for the pH tolerance analysis or supplemented with NaCI to final concentrations: 0%, 0.5%, 1.0% - 9.0% (with 1 % increment) for salinity tolerance analysis. The initial optical density at 600 nm (OD ₆₀ o) was 0.05 in each assay. The cultures were incubated with agitation at 145 rpm at 30°C or other tested temperatures (temperature assay). Catalase activity was determined by the production of bubbles after addition of 3% (v/v) hydrogen peroxide solution. Oxidase activity was tested using discs containing N,N-dimethyl-p-phenylenediamine oxolate and α-naphthol (Sigma-Aldrich, USA). Other biochemical features of the microorganisms were determined using a GEN III Biolog microplate (Biolog, USA) and an API 20NE systems (bioMerieux, France) according to manufacturer's instructions. Degradation of SDS was tested in minimal medium with 0.1 % SDS as a sole carbon source and measured by colorimetric assay, as described in Example 2. Fluorescence pigment production was tested on King B medium (King et al., 1954). As reference strains: Pseudomonas jessenii DSM 17150 ^T (=CIP 105274 ^T ), Pseudomonas baetica DSM 26532 ^T (=390a ^T , =LMG 25716 ^T ), Pseudomonas reinekei DSM 18361 ^T (=MT-1 ^T ), Pseudomonas vancouverensis DSM 17555 ^T and Pseudomonas umsongensis DSM 16611 ^T were used, obtained from the DSMZ collection.

Results of morphological, physiological and biochemical characterisation showed that:

Pseudomonas laurylsulfatovorans AP3_22 deposited in the Polish Collection of Microorganisms (PCM) of the Institute of Immunology and Experimental Therapy PAN at number B/00140 and Pseudomonas jessenii AP3_16 deposited in the Polish Microbiological Collection (PCM) of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences under the number B/00139 belong to Pseudomonas genus. Both strains formed round colonies with smooth shapes and a beige (AP3_22) or milky (AP3_16) color. The cells are Gram-negative bacillus with an average size of 0.6 pm in width and 1.9 pm in length, moving with a single flagella. Cells produce fluorescent pigment on King B medium. Growth is observed in 0-6% NaCI (optimal 0-2%), 5-10 pH (optimal pH 7) and at 8-42°C (optimal 30°C). The strains show a positive result in tests for catalase and oxidase activity, and negative for urease, arginine dehydrolase and β-galactosidase. In contrast to the AP3_16, the AP3_22 strain, similarly to the P. jessenii DSM17150 and P. umsongensis DSM 1661 1 , has the ability to reduce nitrates. The strains do not hydrolyze esculin and gelatin, and do not ferment glucose. The strains assimilate: L-arabinose, potassium gluconate, decanoic acid, malic acid, trisodium citrate and phenylacetic acid, but do not assimilate adipic acid. Complete information on the phenotypic traits obtained in the Biolog GENIII and API20 NE tests are listed in Table 1 , below. The main distinguishing features of the AP3_16 and AP3_22 strains from related Pseudomonas species were: the use of sodium dodecyl sulphate (SDS) as the sole carbon source and the ability to oxidize sucrose, a-keto-butyric acid and acetoacetic acid. The G + C content of the DNA of AP3_22 strain is 59.6 m ^' ol% and AP3_16 strain is 60.1 mol%, wherein the values for reference strains are: P. jessenii DSM 17150 ^T - 59.7; P. baetica DSM 26532 ^T - 58.8; P. reinekei DSM 18361 ^T - 59.2 and P. umsongensis DSM 16611 ^T - 59.7.

Table 1. Physiological and biochemical characteristic of P. jessenii AP3_16, P. laurylsulfatovorans AP3_22 and the other closest Pseudomonas type strains. The strains were tested using Biolog GEN III Microplate and API20 NE strips. Lanes: 1 - P. jessenii AP3_16 (deposited as B/00139); 2 - P. laurylsulfatovorans AP3_22 (deposited as B/00140); 3 - P. jessenii CIP 105724 ^T ; 4 - P. vancouverensis DSM 17555 ^T ; 5- P. umsongensis DSM 16611 ^T ; 6 - P. reinekei DSM 18361 ^T ; 7 - P. baetica DSM26532 ^T ; +, positive; -, negative. Carbon source 1 2 3 4 5 6 7

Dextrin +

D-Maltose

D-Trehalose

D-Cellobiose

Gentiobiose

Sucrose + +

D-Turanose

Stachylose

D-Raffinose

A-D-Lactose

D-Melibiose

B-Methyl-D-Glucoside

D-Salicin

N-Acetyl-D-Glucosamine + + + + - - +

N-Acetyl-B-D-Mannosamine

N-Acetyl-D-Galactosamine

N-Acetyl Neuraminic Acid

A-D-Glucose + + + + + + +

D-Mannose + + + + + + +

D-Fructose + + + + + + +

D-Galactose + + + - + + +

3-Methyl Glucose

D-Fucose + - - + - + +

L-Fucose + - - + - + -

L-Rhamnose

Inosine - - - + - + +

D-Sorbitol

D-Mannitol + - + + - + +

D-Arabitol + - + + - + +

Myo-lnositol

Glycerol + + + + + + +

D-Glucose-6P04

D-Fructose-6-P04 + - - - - + +

D-Aspartic Acid - - + - - + -

D-Serine + + + + - + +

Gelatin

Glycyl-L-Proline - - + - + + -

L-Alanine + + + + + + + Carbon source 1 2 3 4 5 6 7

Carbon source 1 2 3 4 5 6 7

8% NaCI - - - - + + +

1 % Sodium Lactate + + + + + + +

Fusidic Acid + + + + + + +

D-Serine + + + + + + +

Troleandomycin + + + + + + +

Rifamycin Sv + + + + + + +

Minocycline + +

□neomycin + . + + + + + +

Guanidine Hcl + + + + + + +

Niaproof 4 + + + + + + +

Vancomycin + + + + + + +

Tetrazolium Violet + + + + + + +

Tetrazolium Blue + + + + + + +

Nalidixic Acid + + + + + + +

Lithium Chloride + + + + + + +

Potassium Tellurite + + + + + + +

Aztreonam + + + + + + +

Sodium Butyrate + - - - - + -

Sodium Bromate + + - + + + +

API20 NE feature 1 2 3 4 5 6 7

Nitrate Reduction - + + + + - -

Indole Production

Glucose Fermentation

Arginine Dehydroiase - - - - + - +

Urease

Esculin Hydrolysis

Gelatin Hydrolysis + β-Galactosidase

Assimilation of:

Glucose + + + + + + +

Arabinose - + + - + + +

Mannose + + + - + - +

Mannitol + - + + - + +

N-Acetyl-Glucosamine + + + + - - +

Maltose

Potassium Gluconate + + + + + + +

Capric Acid + + + + + + +

Adipic Acid Carbon source 1 2 3 4 5 6 7

Malate + + + + + + +

Trisodium Citrate + + + + + + +

Phenylacetic Acid + + + + + + -

Example 7 - Initial identification of enzymes produced by bacterial strains responsible for the degradation of anionic sufrace active agents.

7.1 Crude cell extracts preparation

Bacterial cells of AP3_10, AP3_16, AP3_19, AP3_20, and AP3_22 strains were precultured overnight in LB medium, centrifuged, inoculated in 100 ml of minimal medium supplemented with 1 g/L SDS or glucose as the carbon source to OD ₆ oo = 0.15, and incubated for 20 h at 30°C with agitation (140 rpm). Cells were harvested by centrifugation at 10,000 g for 15 minutes at RT. Cell pellets were resuspended in lysis buffer (50 mM HEPES, pH 7.5, 300 mM NaCI, 20 mM imidazole, 50 μΜ PMSF, 10 mM β-mercaptoethanol, 0.1 % Tween 20, 10% glycerol, and lysozyme) and additionaly disrupted by sonication (Diagenode sonication system) in a cooled water bath (4°C) at high power (300 W) for 30 cycles of 30 s on and 30 s off. Cell debris was removed by centrifugation at 14,000 g for 30 minutes at 4°C and the supernatants were stored at -20°C.

7.2. Analysis of enzymes with alkylsulfatase activity - non-denaturing polyacrylamide gel Zymography

To determine the sulfatase activity, the crude cell extracts obtained from each of the fast- degrading strains (60 pg of protein) were separated using 8% native polyacrylamide gel (Sambrook and Russel, 2001 ). The electrophoresis was carried out at 100 V at 4°C in 0.378 M Tris-glycine buffer (pH 8.3). After washing in ultrapure water (MiliQ), the gel was incubated in a developing solution (20 mM SDS in 0.1 M Tris-CI pH 7.5) at 30°C. Active alkyl sulfatases presence was visualized by the formation of white bands of insoluble alcohol.

Three isolates, AP3_10, AP3_20, and AP3_22, showed only one band on zymography, suggesting the presence of only one enzyme with , alkyl sulfatase activity toward SDS (Fig. 6). Whereas for AP3_16 and AP3_19, which are phylogenetically distinct from the others, two bands were visible on zymography, suggesting the presence of two different enzymes or two isoforms of alkyl sulfatases.

Example 8 - Genome sequencing of fast SDS-degrading bacterial strains, and identification of potential anionic surface active agents degrading enzymes in genome sequences

8.1. Genomic DNA isolation, library preparation and sequencing For whole genome sequencing of strains AP3_22 ^T and AP3_16 ^T , DNA was isolated following a previously described protocol (Furmanczyk et al., 2017) based on enzymatic lysis with lysozyme, achromopeptidase and proteinase K, followed by phenokchloroform extraction and ethanol precipitation. The isolated genomic DNA was used to prepare two types of libraries for each strain: 1) paired-end library with average insert size 500 bp (using KAPA HTP Library Preparation Kit for lllumina platforms according to manufacturer's protocol) (Kapa Biosystems, USA), 2) Nextera® Mate Pair library with average insert size 8 kbp (using lllumina protocol). The libraries were verified using a 2100 Bioanalyzer (Agilent, USA) High-Sensitivity DNA Assay and KAPA Library Quantification Kit for the lllumina (Kapa Biosystems, USA). High-throughput sequencing was performed using an lllumina MiSeq and dedicated reagent kits (MiSeq Reagent Kit v3, 600 cycles) (lllumina, USA) with read length of 2x 300 bp.

8.2. Genome assembly and annotation of AP3_22 and AP3_16 strains and identification of potential genes involved in the first stage of SDS degradation

Filtrated reads from the sequencing were processed as previously described (Furmanczyk et al., 2017) were used to reconstruct the genomes of the analysed strains. Genomes were assembled using SPAdes 3.9.0 (Bankevich et al., 2012). Obtained nucleotide contigs longer than 1 kbp were annotated using NCBI PGAP (Tatusova et al., 2016). Among the open reading frames, based on sequence similarity (for example using BLAST tools), potential sulfatases that can participate in the degradation of SDS have been identified. Six genes were identified in the genome of the AP3_22 strain, and fifteen complete coding sequences were found in the genome of the AP3_16 strain.

Example 9 - Cloning of identified enzyme into expression vectors. Verification of enzymatic activity (in the presence of native promoter) in a host other than the environmental bacterium

9.1. Cloning of potential sulfatase genes and functionality verification in E. coli

DNA sequences coding potential sulfatases involved in SDS degradation with putative promoter regions (six from the genome of the AP3_22 strain and fifteen from the AP3_16 strain) were amplified using designed primer pairs (Table 2, below). Fragments of the genomes were amplified by PCR. The PCR mixture (50 pL) consisted of: template (genomic DNA of each strain (100 ng/pL) - 1 pL); a dNTP mixture (0.2 mM each); pair of primers (0.2 μΜ each), Phusion High-Fidelity Polymerase DNA Polymerase (0.02 U/pL, Thermo) supplied with 1 x HF-buffer. PCR reaction conditions were: initial denaturation (99°C for 5 minutes), 35 cycles: denaturation (99°C for 30 seconds), annealing (56°C for 30 s), elongation (72°C for 30 - 90 seconds (details in Table 2 below)); and final elongation (72°C for 5 minutes). The PCR products were purified using AMPureXP (Beckman Coulter, United States) with a 0.8:1 ratio, then cloned into pCRTMBIunt ll-TOPO R (Invitrogen, United States) and transformed into E. coli TOP10. The constructs were verified by Sanger sequencing of the inserts and then preliminary tests of the ability to SDS degradation by obtained strains were carried out. The overnight LB-precultures were used to inoculate fresh LB medium (1 :50 v/v) with kanamycin and SDS (1 g/L). After 24 h of incubation (37°C, 140 rpm), the SDS concentration in the media was determined by colorimetric assay using Stains-all reagent.

Table 2. Primers sequences for PCR amplification of genes encoding proteins with potential sulfatase activity.

Summary of the experiment is presented in Fig. 7 and Table 3 below. Two E. coli strains carrying the pCR plasmid with fragments of genomes encoding potential sulfatases: CD175_09595 or B0D71_15760, could completely decompose SDS under the conditions tested in the experiment.

Table 3. Analysis of SDS degradation by E. coli cells transformed with plasmids encoding proteins identified in the genomes of AP3_16 (B/00139) and AP3_22 (B/00140) strains with potential sulfatase activity. accession number SDS [%] standard deviation Automatic protein annotation

CD175_04430 103,09 5,43 arylsulfatase

CD175_07120 97,2 4,88 arylsulfatase

CD175_08470 101 ,1 0,85 arylsulfatase

CD175_08875 97,23 2,38 arylsulfatase

CD175_09340 95,77 2,59 arylsulfatase

CD175_14500 117,94 9,88 arylsulfatase

CD175J4675 100,94 1 ,22 arylsulfatase

CD175J4710 120,09 3,58 arylsulfatase

CD175_27360 97,63 0,84 arylsulfatase

CD175_30210 100,62 2,79 arylsulfatase

CD175_04445 99,85 3,78 hipotetical protein

CD175_30230 103,16 5,05 alkylsulfatase

CD175_09595 0,35 1 ,69 alkyl/arylsulfatase

CD175J 3190 106,49 11 ,05 sulfatase

CD175_22425 11 1 ,23 10,86 sulfatase

B0D71_00255 102,87 3,45 arylsulfatase

B0D71_03695 101 ,66 4,23 arylsulfatase

B0D71_04065 99,68 3,8 arylsulfatase

B0D71J5760 -1 ,27 1 ,06 alkyl/arylsulfatase

B0D71_16010 107,59 1 ,64 arylsulfatase

B0D71_22840 105,69 1 ,15 arylsulfatase

E.coli - negative control 105,93 1 ,95 -

Example 10 - Overproduction, purification and characterization of novel enzymes degrading anionic surface active agents

10.1. Cloning of genes encoding alkylsulfatases into a pET vector

Alkylsulfatase encoding genes identified in Example 9 were amplified using the following primer pairs: A16S14FT

(GTTTAACTTTAAGAAGGAGATATACCATGGATGCCTCGTTTCACCTTGAG) (SEQ ID NO:

53) and A16S14RT (GGTGACCCTGAAAATACAAATTCTCCTCGAGCGGGGTAACGATATTGAACT) (SEQ ID NO:

54) protein CD175_09595 and A22S04FT (GTTTAACTTTAAGAAGGAGATATACCATGGATGCCTCGTTTCACCTTGAGCCCTCG) (SEQ ID NO: 55) and A22S04RT

(GGTGACCCTGAAAATACAAATTCTCCTCGAGAGGCGTGACGATATTGAACTGCG) (SEQ ID NO: 56) for protein B0D71_15760. The PCR mixture (50 pl_) consisted of: template (genomic DNA of each strain (100 ng/pL) - 1 pL); a dNTP mixture (0.2 mM each); primers pair (0.2 pM each), Phusion High-Fidelity Polymerase DNA Polymerase (0.02 U/pL, Thermo) supplied with 1x HF-buffer. PCR reaction conditions were: initial denaturation (99°C for 5 minutes), 10 cycles: denaturation (99°C for 30 seconds), annealing (56°C for 30 s), elongation (72°C for 65 seconds) than 20 cycles: denaturation (99°C for 30 seconds), annealing (62°C for 30 s), elongation (72°C for 65 seconds ); and final elongation (72°C for 5 minutes). The PCR products were purified using AMPureXP (0.8 x PCR product: 1.0 x AMPureXP) according to the manufacturer's recommendations, then cloned using the sequence- and ligation-independent cloning protocol (Jeong, 2012), into the pET28 derived vector that allows obtaining a recombinant protein with a histidine tag (6xHis) at the C-terminus of the protein (with the possibility of tag cleavage with the TEV protease). For this purpose, the vector was digested with restriction enzymes: Ncol and Xhol (FastDigest enzymes, Thermo), and then purified by isolation from agarose gel after electrophoretic separation. The vector (100 ng) was mixed with the insert at a molar ratio of 1 :4 and incubated with the addition of T4 DNA polymerase (0.15 U/pL, New England Biolabs) in 1x NEBuffer 2 buffer (New England Biolabs) supplemented with BSA (0,1 mg/ml) for 2.5 minutes at room temperature. Reactions were stopped by 10-minutes incubation on ice. The mixture was then transformed into E. coli MH1. The plasmids were verified by digestion with restriction enzymes followed by sequencing of the inserts. Positively verified construct was transformed into the BL21 E. coli strain.

10.2. Overproduction and purification of selected alkylsulfatases

The overnight LB-precultures of E. coli BL21 (DE3) strains carrying pET plasmids with ^· cloned genes encoding proteins B0D71_15760 or CD175_09595, were used to inoculate LB medium (1 :50 v/v) with kanamycin. After 2 h of incubation (37°C, 150 rpm), the cultures were moved to 18 °C and IPTG to final concentration of 0.5 mM was added. After 24 h of incubation at 18°C with agitation (150 rpm), the cells were harvested by centrifugation (8000 rpm, 20 min, RT). Cell pellets were resuspended in lysis buffer (50 mM HEPES pH 7.5, 300 mM NaCI, 20 mM imidazole, 50 mM PMSF, 10 mM β-mercaptoethanol, 0.1 % Tween 20, 10% glycerol, supplemented with lysozyme). Cells were lysed by sonication in the Bioruptor Plus sonication system (Diagenode) in a cooled water bath (4°C) at high power (300 W) for 30 cycles of 30 s on and 30 s off. Debris was removed by centrifugation at 4°C (20 min, 14000 x g). The protein was purified using Protino 96 Ni-IDA. Clarified lysate was loaded onto preactivated column. The resin was washed with 50 column volumes of wash buffer I (50 mM HEPES pH 7.5, 300 mM NaCI, 10 mM MgCI ₂ ) and then with 25 column volumes of wash buffer II (50 mM HEPES pH 7.5, 300 mM NaCI). Protein was eluted with three fractions (300 ml each) of elution buffer (50 mM HEPES, 300 mM NaCI, 250 mM imidazole pH 7.5). Fractions containing the purified protein were dialyzed overnight against dialysis buffer (50 mM HEPES, 300 mM NaCI, 10% glycerol, 10 mM β-mercaptoethanol, pH 7.5). Protein concentration was measured using Bio-Rad Protein Colorimetric Assay with BSA as a standard curve.

10.3. Determination of exemplary alkylsulfatases properties

Alkylsulfatase activity test

The activity of each purified proteins: B0D71_15760 and CD175_09595 was assayed by colorimetric method using Stains-all reagent. Two ml_ of enzyme solution (150 mg/mL) was mixed with 100 mL of buffer (50 mM Tris-HCI) containing SDS with a final concentration of 0.01 % (w/v). After incubation at 37°C for 5 min, the reaction was terminated by incubation of the sample at 100°C for 10 min. The SDS concentration was determined in a 96-well plate using colorimetric assay with reference to the standard curve. Eight μΙ_ of the reaction mixture was added to 100 μΐ_ of water and 100 μΙ_ of a working Stains-all solution (0.05 mg/ml Stains-all in a formamide: isopropanol:water (2:1 :37)). The absorbance at 438 nm was measured after 10 minutes of incubation. For both protein preparations, the SDS degradation activity was observed.

Effects of temperature and pH on sufatases activity

The optimal temperature for the activity of the alkylsulfatases: B0D71_15760 and CD175_09595 was determined by carrying out the enzyme activity assay in the range of temperatures (4-90°C) in 50 mM Tris-HCI buffer (pH 8.0) for 5 min. The optimal pH for sulfatases was assayed in a pH range of 4.0 - 10.0 with: citrate buffer for pH 4-5; citrate- phosphate buffer for pH 6; Tris-HCI buffer for pH 7-8 and glycine-NaOH buffer for pH 9-10. The relative activity was defined as the percentage of activity determined with respect to the maximum activity achieved in optimal conditions of each sulfatase.

Alkylsufatases thermostability

The thermostability of the recombinant enzymes: B0D71_15760 and CD175_09595 was determined by measuring the residual activity of the enzyme, exposed to 60 and 70 ^° C for 1 h. The relative activity was calculated with reference to the activity of the enzyme preparation not incubated at elevated temperature.

Effects of various reagents on sulfatases activity

The effect of ions: Ca ²⁺ , Cu ²⁺ , Mg ²⁺ , Mn ²⁺ , Na ⁺ , K ⁺ used as chloride salts, and EDTA on the alkylsulfatases: B0D71_15760 and CD175_09595 activity was tested. The specified concentrations (10 mM and/or 100 mM) of the mentioned reagents were added to the reaction mixture and the enzyme's activity was measured. In each case, the relative activity was defined as the percentage of activity determined in the standard conditions without any additive.

Exemplary alkylsulfatases properties Both enzymes B0D71_15760 and CD175_09595 exhibit activity at a similar pH range (6.0-9.0 pH) with maximum activity at pH 8.0 (Fig. 8A). Also the influence of a broad range of temperatures (30-90°C) was tested. The optimal temperature for CD175_09595 activity was 60°C, whereas for B0D71_15760 it was 70°C (Fig. 8B). The effect of various additives on sulfatase activity is presented below in Table 4. These experiments showed that K ⁺ and Mg ²⁺ (in either 10 mM or 100 mM concentration) increased activity of both tested alkylsulfatases, whereas they are inhibited by Cu ²⁺ , EDTA and Ca ²⁺ in 100 mM concentrations. Analyzed enzymes differ in sensitivity to Na ⁺ , Mn ²⁺ , and 10mM Ca ²⁺ . The activity of B0D71_15760 increased in the presence of 10mM Na ⁺ , however, 100 mM Na ⁺ did not affect enzyme activity. Furthermore, activity of CD175_09595 is inhibited by a higher Na ⁺ concentration, whereas a lower concentration of sodium ions has no effect on the activity of this enzyme. Also, Mn ²⁺ did not influence the CD175_09595 activity, although it had positive effect on B0D71_15760, similar to effect caused by 10 mM Ca ²⁺ .

The thermostability studies showed that the B0D71_15760 protein retains its activity after an hour incubation at 60°C, but not at 70°C. Alkylsulfatase CD175_09595 is sensitive to incubation at both tested temperatures. Both enzymes are inactivated by a 10-minute incubation at 100°C (Table 4).

Table 4. Effect of metal ions, chelators and preincubation in different temperatures on activity of the recombinant B0D71_15760 and CD175_09595 enzymes.

Concent B0D71. J 5760 CD175. 09595

Reagent ration Relative activity Standard Relative activity Standard

(mM) (%) deviation (%) deviation

Ca 10 134.1 9.9 107.6 7.9

Ca 100 65.8 16.6 10.6 8.3

Na 10 132.1 6.5 103.7 5.6

Na 100 108.9 7.3 89.0 4.5

10 . 157.4 6.2 125.1 1.5

K 100 197.4 13.8 132.0 11.5

Mg 10 181.1 8.3 124.4 8.3

Mg 100 158.1 2.1 126.0 4.0

Cu 10 61.0 25.4 48.0 10.0

Mn 10 182.6 14.4 109.4 7.3

EDTA 10 32.4 4.8 33.0 3.7 Concent B0D71. J 5760 CD175. 09595

Reagent ration Relative activity Standard Relative activity Standard

(mM) (%) deviation (%) deviation

B0D71. J5760 CD175. 09595

pre-incubation Relative activity Standard Relative activity Standard

(%) deviation (%) deviation

60°C, 1 h 79.5 15.7 -7.7 8.5

70°C, 1 h -0.2 15.7 3.8 2.9

100°C, 10 min 0.0 4.7 8.5 9.4

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In the following sequence listing:

SEQ ID NR: 1 - corresponds to amino acid sequence of alkylsulfatase B0D71_15760 obtained from AP3_22

SEQ ID NR: 2. - corresponds to amino acid sequence of alkylsulfatase CD175_09595 obtained from AP3_16

SEQ ID NR: 3. - corresponds to nucleotide sequence of the gene encoding alkylsulftase B0D71_15760 obtained from AP3_22

SEQ ID NR: 4. - corresponds to nucleotide sequence of the gene encoding alkylsulftase CD175_09595 obtained from AP3_16

SEQ ID NR: 5 - corresponds to 16S rRNA sequence for Pseudomonas jessenii AP3_16 deposited under number B/00139

SEQ ID NR: 6 - corresponds to 16S rRNA sequence for Pseudomonas laurylosulfatovorans AP3_22 deposited under number B/00140

The electronic version of the sequence list submitted with this application includes a reference to all sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 56 (indicated in the description). The following sequence list contains only references to relevant sequences.

Gly Asn Leu Asp Gin Lys Trp Tyr Thr Arg Gly Tyr Tyr Gly Ser Leu 405 410 415

Ser Phe Asn Thr Arg Ala Val Tyr Gin Arg Tyr Met Gly Phe Tyr Asp

420 425 430

Gly Asn Pro Ala Asn Leu Asn Pro Leu Pro Pro Val Glu Thr Ala Arg

435 440 445

Arg Thr Val Glu Ala Met Gly Gly Glu Ala Ala Val Leu Glu Lys Met 450 455 460

Arg Gly Ala He Thr Gin Gly Asp Tyr Arg Trp Ala Ala Gin Leu Gly 465 470 475 480 Asn Gin Val Leu Phe Ala Asn Pro Asp Asn Gly Asp Ala Arg Lys Thr

485 490 495

Gin Ala Glu Ala Leu Glu Gin Leu Gly Tyr Gin Ser Glu Asn Ala Thr

500 505 510

Trp Arg Asn Met Tyr Leu Thr Gly Ala Met Glu Leu Arg Thr Gly Val

515 520 525

Pro Pro His Ser Gly Ser Ser Val Ser Val Asp Met Val Arg Ala Met 530 535 540

Ser Pro Glu Met Phe Phe Asp Tyr Leu Ala Val Arg Leu Asp Ser Glu 545 550 555 560 Lys Ala Val Gly His Asp Leu Thr Leu Asn Trp Thr Phe Glu Asp Gin

565 570 575

Asn Lys Asp Phe Asn Leu Thr Leu Arg Asn Gly Val Leu Thr His Arg

580 585 590

Thr Gly Leu Asn Pro Gin Ala Asp Ala Gly Val Ser Met Ser Lys Ala

595 600 605

Thr Leu Glu Gin He Ser Leu Lys Gin Leu Asp Phe Pro Thr Ala He 610 615 620

His Lys Gly Leu He Lys Leu Gin Gly Asn Gly Lys Lys Leu Gly Glu 625 630 635 640 Leu Met Ser Ser Leu Asp Thr Phe Ser Pro Gin Phe Asn He Val Thr

645 650 655

Pro

<210> 2

<211> 657

<212> PRT

<213> Pseudomonas spp. (B/00139)

<400> 2

Met Pro Arg Phe Thr Leu Ser Pro Arg Gly Leu Leu Ala Cys Leu He

1 5 10 15

Thr Ala Cys Leu Ala Gin Ala Val Val Ala Ala Asp Ala Pro Val Ala

20 25 ^' 30

Ala Ser Pro Gin Thr Thr Ala Ser Asn Ala Ala Val Leu Lys Gin Leu

^' 35 40 45

Pro Phe Thr Asp Arg. Thr Asp Tyr Glu Ser Val Thr Lys Gly Leu He

50 55 60

Ala Pro Phe Lys Gly Gin Val Lys Asp Ala Ala Gly Lys Val He Trp 65 70 75 80

Asp He Gin Ala Tyr Asp Phe Leu Ala Lys Asp Gin Ala Pro Glu Ser

85 90 95

Val Asn Pro Ser Leu Trp Arg Leu Ala Gin Leu Ser Ala His Ala Gly

100 105 110

Leu Phe Glu Val Ser Pro Arg Leu Tyr Gin Val Arg Gly Leu Asp Leu

115 120 125

Ala Asn Met Thr He He Glu Gly Asp Asp Gly Leu He He He Asp

130 135 140

Pro Leu Thr Met Ala Glu Thr Ala Lys Ala Ala Leu Asp Leu Tyr Tyr 145 150 155 160

Gin Asn Arg Pro His Lys Pro Val Val Ala Val He Tyr Ser His Thr

165 170 175

His Val Asp His Phe Gly Gly Val Arg Gly Val He Asp Glu Ala Asp

180 185 190 SEQUENCE LISTING:

<110> IBB

<120> Decontamination method of sulfur esters polluted environment, new bacterial strains and use of thereof.

<130> PZ/5037 /AGR/PCT

<160> 6

<170> Patentln version 3.5

<210> 1

<211> 657

<212> PRT

<213> Pseudomonas spp. (B/00140)

<400>- 1

Met Pro Arg Phe Thr Leu Ser Pro Arg Gly Leu Leu Ala Cys Leu lie

1 5 10 15

Thr Ala Cys Leu Ala Gin Ser Val Val Ala Ala Asp Ala Pro Val Ala

20 25 30

Ala Ser Ser Gin Thr Thr Ala Ser Asn Ala Ala Val Leu Gin Gin Leu

35 40 45

Pro Phe Thr Asp Arg Thr Asp Phe Glu Ser Val Ser Lys Gly Leu lie

50 55 60

Ala Pro Phe Lys Gly Gin Val Lys Asp Ala Ser Gly Lys Val He Trp

65 70 75 80

Asp He Gin Ala Tyr Asp Phe Leu Ala Lys Asp Lys Ala Pro Asp Ser

85 90 95

He Asn Pro Ser Leu Trp Arg Leu Ala Gin Leu Asn Ala His Ala Gly

100 105 110

Leu Phe Glu Val Ser Pro Arg Leu Tyr Gin Val Arg Gly Phe Asp Leu

115 120 125

Ala Asn Met Thr He He Glu Gly Asp Asp Gly Leu He He He Asp

130 135 140

Pro Leu Thr Val Ala Glu Thr Ala Lys Ala Ala Leu Asp Leu Tyr Tyr

145 150 155 160

Gin Asn Arg Pro His Lys Pro Val Val Ala Val He Tyr Ser His Thr

165 170 175

His Val Asp His Phe Gly Gly Val Arg Gly Val He Asp Glu Ala Asp

180 185 190

Val Lys Ala Gly Lys Val Lys Val Phe Ala Pro Ala Gly Phe Met Glu

195 200 205

His Val Met Ser Glu Asn Val Tyr Ala Gly Thr Ala Met Ser Arg Arg

210 215 220

Ala Gin Tyr Gin Phe Gly Ser Leu Leu Pro Arg Gly Asp His Gly Gin

225 230 235 240

Val Asp Ala Gly Leu Gly Lys Ser Ser Pro Asn Gly Gly Thr Val Thr

245 250 ·■ 255

Leu He Pro Pro Thr Asp Leu He Asp Lys Glu Leu- Glu Thr Arg Thr

260 265 270

He Ala Gly Leu Glu Val Glu Phe Gin Leu Thr Pro Gly Thr Glu Ala

275 280 285

Pro Ala Glu Met Asn Leu Tyr Leu Pro Gin Leu Arg Ala Leu Cys Met

290 295 300

Ala Glu Asn Ala Thr Gin Met Met His Asn He Leu Thr Pro Arg Gly

305 310 315 320

Ala Gin Val Arg Asp Ala Lys Ala Trp Ala Glu Tyr Leu Asp Ser Ser

325 330 335

Leu Ala Arg Tyr Gly Asn Lys Ser Asp Val Leu Phe Ala Gin His Asn

340 345 350

Trp Pro Thr Trp Gly Gly Glu Arg He Arg Thr Phe Leu Ala Asp Gin

355 360 365

Arg Asp Met Tyr Ala Phe Leu Asn Asp Arg Thr Leu His Leu Leu Asn

370 375 380

Gin Gly Leu Thr Pro Leu Glu He Ala Asp Ser He Lys Lys Leu Pro

385 390 395 400 Val Lys Ala Gly Lys Val Lys Val Phe Ala Pro Ala Gly Phe Met Glu

195 200 205

His Val Met Ser Glu Asn Val Tyr Ala Gly Asn Ala Met Ser Arg Arg

210 215 220

Ala Gin Phe Gin Phe Gly Ser Leu Leu Pro Arg Gly Glu Lys Gly Gin

225 230 235 240

Val Asp Ala Gly Leu Gly Lys Ser Thr Pro Ser Gly Gly Thr Val Thr

245 250 255

Leu He Pro Pro Thr Asp Leu He Asp Lys Glu Leu Glu Thr Arg Thr

260 265 270

He Ala Gly Leu Glu Val Glu Phe Gin Leu Thr Pro Gly Thr Glu Ala

275 280 285

Pro Ala Glu Met Asn Leu Tyr Leu Pro Gin Leu Arg Ala Leu Cys Met

290 295 300

Ala Glu Asn Ala Thr Gin Met Met His Asn He Leu Thr Pro Arg Gly

305 310 315 320

Ala Gin Val Arg Asp Ala Lys Ala Trp Ala Glu Tyr Leu Asp Gly Ser

325 330 335

Leu Ala Arg Tyr Gly Asp Lys Ser Asp Val Leu Phe Ala Gin His Asn

340 345 350

Trp Pro Thr Trp Gly Gly Glu Arg He Arg Thr Phe Leu Ala Asp Gin

355 360 365

Arg Asp Met Tyr Ala Phe Leu Asn Asp Arg Thr Leu His Leu Leu Asn

370 375 380

Gin Gly Leu Thr Pro Leu Glu He Ala Asp Ser He Lys Lys Leu Pro

385 390 395 400

Gly Ser Leu Asp Gin Lys Trp Tyr Thr Arg Gly Tyr Tyr Gly Ser Leu

405 410 415

Ser Phe Asn Thr Arg Ala Val Tyr Gin Arg Tyr Met Gly Phe Tyr Asp

420 425 430

Gly Asn Pro Ala Asn Leu Asn Pro Leu Pro Pro Val Glu Thr Ala Lys

435 440 445

Tyr Thr Val Glu Ala Met Gly Gly Glu Ala Ala Val Leu Glu Lys Met

450 455 460

Arg Ala Ala Met Thr Lys Gly Glu Tyr Arg Trp Ala Ala Gin Leu Gly

465 470 475 480

Asn Gin Leu Leu Phe Ala Asn Pro Asp Asn Gly Asp Ala Arg Lys Ala

485 490 495

Gin Ala Glu Ala Leu Glu Gin Met Gly Tyr Gin Ser Glu Asn Ala Thr

500 505 510

Trp Arg Asn Met Tyr Leu Thr Gly Ala Met Glu Leu Arg Asn Gly Val

515 520 525

Pro Pro His Ala Gly Thr Ser Val Ser Val Asp Met Val Arg Ala Met

530 535 540

Ser Pro Gin Met Phe Phe Asp Phe Leu Ala Val Arg Leu Asp Ser Glu

545 ^' 550 555 560

Lys Ala Ala Gly His Asp Leu Thr Leu Asn Trp Thr Phe Glu Asp Leu

565 570 575

Asn Lys Asp Phe Asn Leu Thr Leu Arg Asn Gly Val Leu Thr His Arg

580 585 590

Ala Gly Leu Asn Ala Gin Ala Asp Ala Gly Val Thr Met Ser Lys Ala

595 600 605

Thr Leu Glu Gin He Ser Leu Lys Gin Leu Asp He Pro Thr Ala He

610 615 620

Gin Lys Gly Leu He Lys Leu Gin Gly Asn Gly Lys Lys Leu Gly Glu

625 630 635 640

Leu Met Thr Ser Leu Asp Thr Phe Ala Pro Gin Phe Asn He Val Thr

645 650 655

Pro

<210> 3

<211> 1974

<212> DNA <213> Pseudomonas spp. (B/00140)

<400> 3

atgcctcgtt tcaccttgag ccctcgtggg ctgctcgctt gcctgatcac cgcctgcctg 60 gcgcaatcgg tagtcgccgc cgatgcaccg gtcgcggcca gctcgcagac cacggcgagt 120 aacgccgccg tgctgcaaca actgcccttc accgaccgca ccgactttga atcggtctcc 180 aagggtttga tcgcgccgtt caagggccag gtcaaggacg cctcgggcaa ggtcatctgg 240 gatatccagg cctacgattt cctcgccaaa gacaaggccc cggattcgat caacccgagc 300 ctgtggcgcc tggcccagct caacgcccat gccggattgt tcgaagtcag cccgcggttg 360 tatcaggtgc gcggcttcga cctggcgaac atgaccatca tcgaaggcga tgacgggctg 420 atcatcatcg acccgctgac agtggccgaa accgccaagg ccgcactgga cctgtactac 480 cagaaccgcc cgcacaaacc ggtggtggcg gtgatctaca gccacaccca cgtcgaccac 540 ttcggcggcg tgcgcggcgt gatcgacgag gccgacgtca aggccggcaa ggtcaaggtg 600 ttcgctcccg ccggcttcat ggaacacgtg atgagcgaga acgtctacgc cggcaccgcc 660 atgagccgcc gcgcgcaata ccagttcggc agcctgctgc cccgtggcga tcatggccag 720 gtcgatgccg gcctgggcaa aagctcgccc aatggcggca ccgtcaccct gatcccgccc 780 accgacctga tcgacaagga actggaaacc cgcaccatcg ccggcctcga ggtggaattc 840 cagctgaccc cgggcaccga ggcaccggcg gaaatgaacc tgtacctgcc gcaactgcgt 900 gccttgtgca tggcggaaaa cgccacgcaa atgatgcaca acatcctcac ccctcgcggt 960 gcgcaagtgc gtgatgccaa ggcctgggcg gagtacctgg acagcagcct ggcgcgttac 1020 ggcaacaaga gcgacgtgtt gttcgcccag cacaactggc cgacctgggg cggcgaacgc 1080 atccgcacct tcctcgccga ccaacgggac atgtacgcct tcctcaatga ccgcaccttg 1140 cacctgctga accagggcct gacgccactg gaaatcgccg actcgatcaa gaaactgccc 1200 ggcaacctgg accagaagtg gtacacccgt ggctactacg gctcgctgag tttcaacacc 1260 cgcgcggtgt atcagcgcta catgggtttc tatgacggca acccggccaa cctcaatccg 1320 ctgccacccg tggaaacagc ccggcgcacg gtcgaggcca tgggcggtga agcggcggtg 1380 ctggagaaaa tgcgtggggc gatcacccag ggtgactacc gctgggcggc gcaactgggc 1440 aatcaagtac tgttcgccaa ccccgacaac ggcgatgcgc gcaaaaccca ggccgaggcc 1500 ctggagcaac tgggttatca aagcgaaaac gccacctggc gcaacatgta cctgaccggt 1560 gccatggaac tgcgcaccgg cgtgccgccg cactctggca gttcggtgtc ggtggacatg 1620 gtgcgggcga tgagcccgga gatgttcttc gactacctgg cggttcgcct ggacagtgaa 1680 aaggccgtgg gccatgacct gaccctgaac tggaccttcg aggaccagaa caaggacttc 1740 aacctgaccc ttcgcaatgg cgtgctgacc caccgcaccg ggctcaatcc ccaggcggat 1800 gccggcgtga gcatgagcaa ggcaaccctg gagcagatca gcctcaagca actggacttc 1860 ccgacggcga tccacaaagg cctgatcaag t _. tgcaaggca atggcaagaa gcttggggag 1920 ttgatgagca gcctcgatac cttttcgccg cagttcaata tcgtcacgcc ttga 1974

<210> 4

<211> 1971

<212> DNA

<213> Pseudomonas spp. (B/00139)

<400> 4

atgcctcgtt tcaccttgag ccctcgtggg ctgctcgctt gcctgatcac tgcctgcctg 60 gcgcaagcgg tggtcgccgc cgatgcaccg gtcgcggcca gcccgcaaac cacggccagt 120 aacgccgccg tcctgaagca actgcccttc accgaccgca ccgactacga gtcggtcacc 180 aagggcttga tcgcgccctt caaaggccag gtcaaggacg ccgcgggcaa ggtcatctgg 240 gatatccagg cctatgactt cctcgccaaa gaccaggccc cggagtcggt caacccgagc 300 ctctggcgcc tggcccaact cagcgcccat gccggtctgt tcgaagtcag ccctcggctg 360 tatcaggtgc gcggcctgga cctggcgaac atgaccatca tcgaaggcga cgacgggctg 420 atcatcatcg acccgttgac catggccgaa accgccaagg cagcactgga cctgtactac 480 cagaaccgac cgcacaaacc ggtggtggcg gtgatctaca gccacaccca cgtcgaccac 540 ttcggtggcg tgcgcggggt catcgacgaa gccgacgtca aggccggcaa ggtcaaagtg 600 ttcgcccccg ccgggttcat ggaacacgtg atgagcgaija,, acgtctacgc tggcaacgcc 660 atgagccgcc gtgcgcagtt ccagttcggc agcctgctgc cccgtggcga aaaaggccag 720 gtcgatgccg gcctgggcaa gagcacgccg agcggcggca ccgtcaccct gatcccgccc 780 accgacctga tcgacaaaga actggagacc cgcaccatcg ccggcctcga ggtggaattc 840 cagttgaccc cgggcaccga ggcgcccgcg gaaatgaacc tgtacctgcc gcaattgcgt 900 gccttatgca tggcggaaaa cgccacgcaa atgatgcaca acatcctcac cccgcgcggc 960 gcgcaagtgc gggacgccaa ggcctgggcc gagtacctgg acggcagcct ggcgcgatac 1020 ggcgacaaga gcgacgtact gttcgcgcag cacaactggc cgacctgggg cggcgaacgc 1080 atccgcacct tcctcgccga ccagcgcgac atgtacgcct tcctcaatga ccgcaccctg 1140 cacctgctga accagggcct gacgccgctg gaaatcgccg actcgatcaa gaaactgccc 1200 ggcagcctgg accagaagtg gtacacccgc ggctactacg gctcgctgag cttcaacacc 1260 cgtgcggtgt atcagcgtta catgggtttc tatgacggca acccggccaa cctcaatccg 1320 ctgccacctg tggaaacggc gaagtacacg gtcgaggcca tgggtggcga agcggcggta 1380 ctggagaaaa tgcgcgcggc gatgaccaag ggtgagtacc gctgggccgc gcaactgggc 1440 aatcaattgc tgttcgccaa cccggacaac ggcgatgcgc gcaaagctca ggctgaggcg 1500 ctggagcaga tgggctatca aagtgaaaac gccacctggc gcaacatgta cctgaccggc 1560 gccatggaac tgcgcaacgg cgtaccgccc catgcgggca cttcggtgtc ggtggacatg 1620 gtgcgggcga tgagcccgca gatgttcttc gacttcctgg ccgtgcgtct ggacagcgaa 1680 aaggccgcag gccatgacct gaccctgaac tggaccttcg aggacctgaa caaggacttc 1740 aacctgaccc tgcgcaacgg cgtgctgacc caccgcgccg gcctcaacgc ccaggcggat 1800 gccggcgtga ccatgagcaa ggccaccctg gaacagatca gcctcaagca actggatatc 1860 cccacggcga tccagaaagg cctgatcaag ttgcaaggca atggcaagaa gctcggggag 1920 ttgatgacca gcctcgatac gtttgcgccg cagttcaata tcgttacccc g 1971

<210> 5

<211> 1532

<212> DNA

<213> Pseudomonas spp. (B/00139)

<400> 5

tgaagagttt gatcatggct cagattgaac gctggcggca ggcctaacac atgcaagtcg 60 agcggatgac aggagcttgc tcctgaattc agcggcggac gggtgagtaa tgcctaggaa 120 tctgcctggt agtgggggac aacgtttcga aaggaacgct aataccgcat acgtcctacg 180 ggagaaagca ggggaccttc gggccttgcg ctatcagatg agcctaggtc ggattagcta 240 gttggtgagg taatggctca ccaaggcgac gatccgtaac tggtctgaga ggatgatcag 300 tcacactgga actgagacac ggtccagact cctacgggag gcagcagtgg ggaatattgg 360 acaatgggcg aaagcctgat ccagccatgc cgcgtgtgtg aagaaggtct tcggattgta 420 aagcacttta agttgggagg aagggcatta acctaatacg ttagtgtttt gacgttaccg 480 acagaataag caccggctaa ctctgtgcca gcagccgcgg taatacagag ggtgcaagcg 540 ttaatcggaa ttactgggcg taaagcgcgc gtaggtggtt cgttaagttg gatgtgaaag 600 ccccgggctc aacctgggaa ctgcattcaa aactgacgag ctagagtatg gtagagggtg 660 gtggaatttc ctgtgtagcg gtgaaatgcg tagatatagg aaggaacacc agtggcgaag 720 gcgaccacct ggactgatac tgacactgag gtgcgaaagc gtggggagca aacaggatta 780 gataccctgg tagtccacgc cgtaaacgat gtcaactagc cgttgggagc cttgagctct 840 tagtggcgca gctaacgcat taagttgacc gcctggggag tacggccgca aggttaaaac 900 tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 960 gcgaagaacc ttaccaggcc ttgacatcca atgaactttc cagagatgga ttggtgcctt 1020 cgggaacatt gagacaggtg ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt 1080 taagtcccgt aacgagcgca acccttgtcc ttagttacca gcacgttatg gtgggcactc 1140 taaggagact gccggtgaca aaccggagga aggtggggat gacgtcaagt catcatggcc 1200 cttacggcct gggctacaca cgtgctacaa tggtcggtac agagggttgc caagccgcga 1260 ggtggagcta atcccacaaa accgatcgta gtccggatcg cagtctgcaa ctcgactgcg 1320 tgaagtcgga atcgctagta atcgcgaatc agaatgtcgc ggtgaatacg ttcccgggcc 1380 ttgtacacac cgcccgtcac accatgggag tgggttgcac cagaagtagc tagtctaacc 1440 ttcgggagga cggttaccac ggtgtgattc atgactgggg tgaagtcgta acaaggtagc 1500 cgtaggggaa cctgcggctg gatcacctcc tt 1532

<210> 6

<211> 1533

<212> DNA

<213> Pseudomonas spp. (B/00140)

<400> 6

ctgaagagtt tgatcatggc tcagattgaa cgctggcggc aggcctaaca catgcaagtc 60 gagcggatga caggagcttg ctcctgaatt cagcggcgga cgggtgagta atgcctagga 120 atctgcctgg tagtggggga caacgtttcg aaaggaacgc taataccgca tacgtcctac 180 gggagaaagc aggggacctt cgggccttgc gctatcagat gagcctaggt cggattagct 240 agttggtgag gtaatggctc accaaggcga cgatccgtaa ctggtctgag aggatgatca 300 gtcacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg gggaatattg 360 gacaatgggc gaaagcctga tccagccatg ccgcgtgtgt gaagaaggtc ttcggattgt 420 aaagcacttt aagttgggag gaagggcagt aaattaatac tttgctgttt tgacgttacc 480 gacagaataa gcaccggcta actctgtgcc agcagccgcg gtaatacaga gggtgcaagc 540 gttaatcgga attactgggc gtaaagcgcg cgtaggtggt ttgttaagtt ggatgtgaaa 600 gccccgggct caacctggga actgcattca aaactgacaa gctagagtat ggtagagggt 660 ggtggaattt cctgtgtagc ggtgaaatgc gtagatatag gaaggaacac cagtggcgaa 720 ggcgaccacc tggactgata ctgacactga ggtgcgaaag cgtggggagc aaacaggatt 780 agataccctg gtagtccacg ccgtaaacga tgtcaactag ccgttgggag ccttgagctc 840 ttagtggcgc agctaacgca ttaagttgac cgcctgggga gtacggccgc aaggttaaaa 900 ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa 960

cgcgaagaac cttaccaggc cttgacatcc aatgaacttt ccagagatgg attggtgcct 1020 tcgggaacat tgagacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg 1080 ttaagtcccg taacgagcgc aacccttgtc cttagttacc agcacgtcat ggtgggcact 1140 ctaaggagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaag tcatcatggc 1200 ccttacggcc tgggctacac acgtgctaca atggtcggta cagagggttg ccaagccgcg 1260 aggtggagct aatcccataa aaccgatcgt agtccggatc gcagtctgca actcgactgc 1320 gtgaagtcgg aatcgctagt aatcgcgaat cagaatgtcg cggtgaatac gttcccgggc 1380 cttgtacaca ccgcccgtca caccatggga gtgggttgca ccagaagtag ctagtctaac 1440 cttcgggagg acggttacca cggtgtgatt catgactggg gtgaagtcgt aacaaggtag 1500 ccgtagggga acctgcggct ggatcacctc ctt 1533